CN102836425B - The Vaccinum Calmette-Guerini of antigen expressed during comprising the latent infection stage - Google Patents
The Vaccinum Calmette-Guerini of antigen expressed during comprising the latent infection stage Download PDFInfo
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- CN102836425B CN102836425B CN201210332759.0A CN201210332759A CN102836425B CN 102836425 B CN102836425 B CN 102836425B CN 201210332759 A CN201210332759 A CN 201210332759A CN 102836425 B CN102836425 B CN 102836425B
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- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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Abstract
The present invention relates to the Vaccinum Calmette-Guerini of antigen expressed during comprising the latent infection stage, be specifically related to a kind of immunogenic composition, vaccine or pharmaceutical composition, it comprises: polypeptide Rv1284 or containing with the immunogenic polypeptide of Rv1284 at least 80% sequence iden.
Description
The application is the applying date is on June 20th, 2006, and application number is 200680022323.4, and denomination of invention is the divisional application of the Chinese patent application of " Vaccinum Calmette-Guerini of antigen expressed during comprising the latent infection stage ".
Technical field
The invention discloses the antigen of hungry induction, or based on the new fused polypeptide deriving from the immunogenic polypeptide of the polypeptide of mycobacterium tuberculosis of inducing between hunger period, the antigen of one or more fused polypeptide of the present invention or hungry induction is for the preparation of the purposes being used for giving the immunogenic composition of people/animal, vaccine or pharmaceutical composition, and such immunogenic composition, vaccine or pharmaceutical composition.
Background technology
People's pulmonary tuberculosis caused by mycobacterium tuberculosis (M.tuberculosis) is a serious global health problem, and according to the data of WHO, it causes annual about 3 million peoples dead.During the twentieth century sixties and the seventies, there is case and decline in the pulmonary tuberculosis (TB) that the whole world is new, but in recent years, part is due to the appearance of the arriving of AIDS and resistance to polybasic drugs (multidrug) bacterial strain of mycobacterium tuberculosis, and obvious change has occurred this trend.
The vaccine being uniquely available for Clinical practice is at present BCG(bacillus calmette-guerin vaccine), effect still some dispute of this vaccine.BCG is the acquisition resistance of induced high levels in TB animal model usually, and in crowd, gives anti-Spread type tuberculosis as meningitis (meningitis) and miliary tuberculosis protection.When giving low age child BCG, it can play antiphthisic protective effect within several years, but effect changes afterwards.More different control experiments (controlled trial), discloses the Vaccine effectiveness of BCG in adult and changes significantly to the scope of validity of 80% protection invalid.This makes to develop and new becomes very urgent thing with the against mycobacterium tuberculosis vaccine of improvement, and it is given very high priority by the World Health Organization (WHO).
Had the trial of much setting forth protectiveness mycobacteria material, different researcheres has reported the resistance obtaining raising after tentative vaccination.Mycobacterium tuberculosis has and secretes some potential protein being suitable for producing new mtb vaccine.The search of candidate molecules is mainly concentrated on to the protein discharged from the antibacterial (dividing bacteria) distinguished.Although a large amount of this protein is characterized, wherein only having minority in animal model, be proved immunne response as subunit vaccine eliciting protective, is wherein ESAT-6 and Ag85B(Brandt etc. the most significantly, 2000).But, also there is no the example (demonstration) of the particular long protective immune response that there are BCG potentiality or strengthen the ability in BCG prophylactic immunization people.The BCG of BCG is used to strengthen not having effect [Colditz, 1994] at most.Although do not have anything to improve significantly with being used alone compared with BCG, carry out BCG booster immunization with Ag85a in inbred mouse strain after or can draw and play a protective role (the IAI2001 such as Brooks; WO0204018).Due to BCG need to distinguish and secretory protein with the immunne response of eliciting protective, therefore the shortage of reinforced immunological effect is mainly because the sensitivity of environment mycobacteria or the residual immunity inoculated from first BCG are replied.Both all cause the acute immune of anti-BCG to reply and thus to the quick suppression of growth, and the removing to BCG.
The process that mycobacterium tuberculosis infects mainly experiences three periods.In acute stage, antibacterial breeds in organ, until immunne response strengthens.The adjustment of the CD4T cell mediated infection of specific sensitization, most important reporter molecule is IFN-γ (IFN-γ) seemingly.Bacterial load (bacterial load) start reduce, bacterial load stable maintenance in low-level time formed incubation period.In this period, mycobacterium tuberculosis is tending towards dormancy by the propagation enlivened, and substantially changes not replicated state into and maintains in granuloma.In some cases, infect and be tending towards reactivation period, the antibacterial of dormancy restarts to copy.Disclose mycobacterium tuberculosis change (Honer zu Bentrup, 2001) along with gene expression from primary infection to preclinical transformation.Also likely change in the antigen-specificity of immunne response, because between the active tour copying to dormancy, antibacterial regulates gene expression.Control all characteristics of latent infection immunne response and cause the factor of reactivation to be unknown substantially.But, there are some evidences to prove relevant with the transformation dominated in cell (dominant cell) type.Although the infection control of cd4 t cell to acute stage is necessary with enough, it is even more important in incubation period that research discloses cd8 t cell reaction.1998, Cole etc. disclosed the complete genomic sequence of mycobacterium tuberculosis, and prediction wherein exists about 4000 open reading frame, discloses the protein sequence (Cole etc., 1998) of nucleotide sequence and presumption.But importantly, this sequence information can not be used for predicting whether this DNA is translated in vivo and is expressed as albumen.As everyone knows, some genes of mycobacterium tuberculosis are up-regulated under the preclinical condition of simulation.But, the finite subset of gene expression total during these are latent infection.In addition, those skilled in the art will easily understand, and a kind of expression of gene is also not enough to become a kind of good candidate vaccine.Determine that whether a kind of albumen be prepare this given albumen by the unique method of immune system recognition during mycobacterium tuberculosis latent infection, and according to suitable test described herein, this albumen is tested.Some protein particular importances also potentially become late antigen (antigen identified during latent infection), this is due to after infection, their major parts have expressed the relatively long time, and now immune system has started initial adaptability defence, and environment also becomes more hostile to mycobacteria.The condition of culture of the hypoxia in vitro of simulated hypoxia pressure has appeared relevant to this respect before this, has been used to now the change of analyzing gene expression aspect.Have been found that and can induce under these conditions or just regulate some antigens significantly, such as 16kDa antigen alpha-crystal albumen (α-crystalin) (Sherman, 2001), Rv2660c and Rv2659c(Betts, 2002) (our application).Another likely interested especially environmental stimulus be hungry, its design reflects that nutrient is limited to granuloma interior (site of latent infection) and gene expression product is just regulated under hunger, therefore likely becomes the interested especially antigen target infected in incubation period.
To express and in the antigen tested as vaccine, the antigen expression being less than half-dozen goes out obvious potentiality in the primary infection phase known more than 20000 kinds.The institute had as therapeutic vaccine is potential (Lowrie, 1999) only to have a kind of antigen to be proved up to now.But this vaccine only works when using as DNA vaccination and is proved to be controversial, program inoculation is used even to make disease progression (Turner, 2000) induction of nonspecific protection because there is other group to claim.On the contrary, as shown in the embodiment provided, use the vaccination technology of best identified, the fused polypeptide that the present invention describes can be impregnated in vaccine.
Further, due to TB vaccine do not cause bactericidal immune but infection control in subclinical (subclinical) level (thus causing the establishment of latent infection subsequently), therefore the invention describes many phases (multiphase) vaccine of the ingredient combination by having prevention and therapy activity.After conventional prophylactic immunization, developing subsequently of the escape (evasion) of primary immune response and latent disease is perhaps the change of antigenic profile (antigenic profile) due to invasion and attack antibacterial at least in part.Therefore, inoculate with the antigen relevant to the TB that hides, should be able to prevent or reduce the formation of latent infection, and therefore will be mixed with the vaccine of antibacterial antigen expressed between the first exponential phase and fallow stadium, during as preventative vaccine, long-term immunity can be improved.Because this vaccine of many phases obviously also can as effectively treating vaccine, the most people that therefore can solve the third world that will accept following TB vaccine may by the problem infected with hiding.
Summary of the invention
The present invention relates to for prevention or/and the immunogenic composition of the infection for the treatment of caused by the species of mycobacterium tuberculosis complex (complex) (mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium africanum etc.), vaccine or pharmaceutical composition (comprising booster shot vaccine and vaccine of many phases), this immunogenic composition, vaccine or pharmaceutical composition contain the antigen of hungry induction or comprise the fused polypeptide of one or more hungry antigen of mycobacterium tuberculosis of inducing, and wherein the unit of fused polypeptide is antigen of mycobacterium tuberculosis.The invention still further relates to the nucleotide sequence of such a fused polypeptide and this fused polypeptide of coding.Further, the present invention relates to by synthesizing or recombinate folded (multiple) peptide of short weight prepared or the purposes growing overlapping (multiple) peptide or nonoverlapping (multiple) peptide.Further, the present invention relates to the antigen of hungry induction or fused polypeptide sequence of the present invention or the nucleotide sequence purposes for the preparation of described immunogenic composition, vaccine or pharmaceutical composition, and the vaccine prepared by the method or pharmaceutical composition.Further, the present invention relates to the vaccine of the antigen or fused polypeptide sequence or nucleotide sequence comprising the induction of the present invention's hunger for the preparation of the purposes of described immunogenic composition, vaccine or pharmaceutical composition, described vaccine gives with BCG simultaneously, also can mix from BCG or in different position or individually dosed with different approach.Further, the present invention relates to and comprise the hungry antigen of induction or the vaccine of fused polypeptide sequence or the nucleotide sequence purposes as BCG Booster.Further, by antigen that is early stage during comprising natural infection and that all express late period, vaccine resists pathogen by causing the immunne response of two steps to make immune system, and no matter this pathogen is with which kind of the most effective epi-position on certain time point comprised between incubation period.
Invention is carefully stated
The invention discloses the immunogenic composition of the fused polypeptide of the antigen comprising hungry induction or the antigens containing one or more hungry inductions, vaccine or pharmaceutical composition.
Aminoacid and the nucleotide sequence of these hungry induction (just regulating more than 6.5 times between hunger period or be connected with the gene inheritance that hunger is induced) antigens appear in sequence table as shown below:
| The antigen of hungry induction | DNA SEQ ID NO | aa SEQ ID NO |
| Rv2655 | 1 | 2 |
| Rv2656 | 3 | 4 |
| Rv2657 | 5 | 6 |
| Rv2658 | 7 | 8 |
| Rv2659c | 9 | 10 |
| Rv2660c | 11 | 12 |
| Rv2661 | 13 | 14 |
| Rv2662 | 15 | 16 |
| Rv2663 | 17 | 18 |
| Rv0188 | 19 | 20 |
| Rv3290c | 21 | 22 |
| Rv3289c | 23 | 24 |
| Rv2034 | 25 | 26 |
| Rv2169c | 27 | 28 |
| Rv0116c | 29 | 30 |
| Rv2558 | 31 | 32 |
| Rv1152 | 33 | 34 |
| Rv3291c | 35 | 36 |
| Rv1284 | 37 | 38 |
| Rv1954c | 39 | 40 |
| Rv3810 | 41 | 42 |
| Rv2517c | 43 | 44 |
| Rv3288c | 45 | 46 |
| Rv0789c | 47 | 48 |
| Rv1955 | 49 | 50 |
| Rv3735 | 51 | 52 |
| Rv3675 | 53 | 54 |
| Rv2270 | 55 | 56 |
| Rv2050 | 57 | 58 |
| Rv3287c | 59 | 60 |
| Rv2557 | 61 | 62 |
| Rv0122 | 63 | 64 |
| Rv2497c | 65 | 66 |
| Rv1250 | 67 | 68 |
| Rv1552 | 69 | 70 |
| Rv2526 | 71 | 72 |
| Rv1809 | 73 | 74 |
| Rv0918 | 75 | 76 |
| Rv0516c | 77 | 78 |
| Rv2745c | 79 | 80 |
| Rv1472 | 81 | 82 |
| Rv1660 | 83 | 84 |
| Rv2302 | 85 | 86 |
In current context, be defined by " unit " of fused polypeptide based on each immunogen polypeptide deriving from mycobacterium tuberculosis polypeptides.Described fusion can comprise 2,3,4,5,6,7,8,9 and even 10 different unit.
In fused polypeptide, the order of unit can be any combination.In ordinal term, the fused polypeptide of all above-mentioned antigen of any combination all belongs within scope of the present invention.Fused polypeptide of the present invention can be used for preparing immunogenic composition, vaccine or pharmaceutical composition, and the BCG be especially described in more detail below strengthens vaccine.
The preferred polypeptide forming fused polypeptide unit together with hungry polypeptide has following Sanger identification number and aminoacid sequence:
| Usually the title of (trivial) | Sanger ID |
| ESAT6 | Rv3875 |
| TB10.4 | Rv0288 |
| Ag85A | Rv3804c |
| Ag85B | Rv1886c |
| ORF2c | Rv3871(C end) |
| TB13.0 | Rv1036 |
| TB9.56 | Rv0285 |
| TB9.8 | Rv0287 |
Preferred fused polypeptide compound comprises following with the polypeptide of different sequence of unit combination, described polypeptide has the antigen (X) of one or more hungry inductions: ESAT6-Ag85A-X, ESAT6-Ag85B-X, Ag8A-X, Ag85B-X, TB10-Ag85A-X, TB10-Ag85B-X, wherein X is antigen of any one hungry induction, and the order of antigen unit can be any one combination, and such as order is wherein contrary or X is placed in middle etc.
But fused polypeptide can build according to one or more hungry antigens of induction and other combination of any one of one or more antigen of mycobacterium tuberculosis.
The analog of fused polypeptide and the nucleotide sequence of this peptide species of encoding all are within the scope of the present invention, and described analog has to be had the aminoacid sequence of at least 80% sequence iden with any part of any one fused polypeptide of the present invention and have immunogenicity.This analog is included in term " polypeptide of the present invention " or " fused polypeptide of the present invention ", and these terms are used alternatingly by description and claims.According to term " nucleotide sequence of the present invention ", it refers to the nucleotide sequence of this peptide species of coding.Further, also comprise folded (multiple) peptide of short weight within the scope of the invention or grow overlapping (multiple) peptide or nonoverlapping (multiple) peptide, described polypeptide has to be had the aminoacid sequence of 80% sequence iden with any one fused polypeptide of the present invention and has immunogenicity.
The present invention strengthens the vaccine in first BCG vaccination immunity, that is, this vaccine gives previous vaccinated BCG individuality.
First aspect of the present invention comprises antigen or the variant of fused polypeptide of above-mentioned hunger induction, its by esterified (lipidated) to provide self auxiliary (self-adjuvating) effect of polypeptide.
Subject immunogenic compositions, vaccine or pharmaceutical composition can pass through as the mucosa delivery of per os, nose, cheek or intramuscular administration, the intradermal administration of routine, by subcutaneous administrations or percutaneous dosing, or any administration that other is applicable to, such as rectally.
In another embodiment, the invention discloses antigen or the purposes of fused polypeptide in the preparation of immunogenic composition, vaccine or pharmaceutical composition of hungry induction as defined above, described immunogenic composition, vaccine or pharmaceutical composition energy and BCG vaccine one are used from prophylactic immunization, booster shot or the inoculation of being used for the treatment of property, resist by the infection of virulent mycobacteria caused by mycobacterium tuberculosis, mycobacterium africanum, Mycobacterium bovis, Mycobacterium leprae or mycobacterium buruli.
In second, the invention discloses immunogenic composition, vaccine or pharmaceutical composition, they comprise the coding as defined above hungry antigen of induction or the nucleotide sequence of fused polypeptide, or comprise can under strict conditions with the nucleotide sequence of the complementation of nucleic acid array hybridizing of the present invention.
Described nucleic acid fragment is preferably DNA fragmentation.Described fragment can be used as medicine discussed below.
In one embodiment, the invention discloses immunogenic composition, vaccine or pharmaceutical composition, they comprise the nucleic acid fragment of the present invention of optionally insertion vector.After the vaccine causing the animal body endoantigen comprising people to express is comprised the animal of people, the resistance strengthened in fact can be given animal and be comprised people, to strengthen the resistance lungy to being caused as M. tuberculosis, mycobacterium africanum, bacillus tuberculosis bovis, Mycobacterium leprae or mycobacterium buruli by virulent mycobacteria by the antigen levels of expressing effectively.
In a further embodiment, the invention discloses the purposes of a kind of immunogenic composition, vaccine or pharmaceutical composition comprising nucleic acid fragment of the present invention for the anti-therapeutic vaccine lungy caused by virulent mycobacteria.
In another further embodiment, the invention discloses immunogenic composition, vaccine or pharmaceutical composition, they can be used to prophylactic immunization together with BCG, or the people of inoculated BCG before giving as reinforcement vaccine, people's opposing is comprised by virulent mycobacteria as mycobacterium tuberculosis with immune animal, mycobacterium africanum, bacillus tuberculosis bovis, Mycobacterium leprae or the tuberculosis caused by mycobacterium buruli, described immunogenic composition, vaccine or pharmaceutical composition comprise non-pathogenic microorganism as effective ingredient as cattle pox, adenovirus or bacillus tuberculosis bovis BCG, wherein, the DNA fragmentation comprising at least one copy of the DNA sequence of above-mentioned fused polypeptide of encoding can express the mode of secreting this fused polypeptide with selectivity to make microorganism, be introduced in microorganism and (be such as placed in plasmid or genome).
In another embodiment, the invention discloses the infectious expression vector containing nucleic acid fragment of the present invention, such as cowpox, adenovirus or bacillus tuberculosis bovis BCG, and the cell of conversion containing carrier described at least one.
In the 3rd, the invention discloses and people is comprised to animal carry out immunity or strengthen their methods to the immunity lungy that virulent mycobacteria causes, wherein virulent mycobacteria is as mycobacterium tuberculosis, mycobacterium africanum, Mycobacterium bovis, Mycobacterium leprae or mycobacterium buruli, and the method comprises and gives animal fused polypeptide defined above, immunogenic composition of the present invention or vaccine of the present invention.
In the 4th, the invention discloses the method being used for the treatment of and suffering from animal lungy (comprising people) that is active or that hide, described tuberculosis is by virulent mycobacteria as mycobacterium tuberculosis, mycobacterium africanum, Mycobacterium bovis, Mycobacterium leprae or mycobacterium buruli cause, and the method comprises and gives animal immunogenic composition defined above, vaccine or pharmaceutical composition.
In the 5th, the antigen or fused polypeptide or the nucleic acid fragment that the invention discloses hunger induction defined above are preparing the purposes in immunogenic composition, vaccine or pharmaceutical composition, and this immunogenic composition, vaccine or pharmaceutical composition are combined with mycobacterium bovis BCG for prophylactic immunization (comprising booster shot) or Therapeutic Vaccination to resist by the infection of virulent mycobacteria caused by mycobacterium tuberculosis, mycobacterium africanum, Mycobacterium bovis, Mycobacterium leprae or mycobacterium buruli.
Vaccine of the present invention, immunogenic composition, vaccine and pharmaceutical composition can by prophylactically for not infecting the experimenter of virulent mycobacteria, or cross the individuality of mycobacterium tuberculosis BCG for previous vaccination, or the experimenter of therapeutic ground for infecting virulent mycobacteria.
Be appreciated that embodiment immunogenic polypeptide as described also whole other side used in the present invention of the present invention first aspect; Vice versa.
Whole through description, unless the context requires otherwise, otherwise word " comprises/comprises (comprise) " or its change saying as " comprise/comprise (comprises) " or " comprise/comprise (comprising) " by be understood as that include as described in composition or overall or the group of composition or the group of entirety the meaning, but itself and non-excluded any other composition or the overall or group of composition or the group of entirety.
Definition
Hungry
According to term " hunger ", it is understood to make organism to lose carbon, nitrogen or the energy, their any one combination and even all.
The albumen of hungry induction
According to term " albumen of hungry induction ", it is understood as that mycobacteria is after hunger is coerced, and is transcribing or protein level is induced any one protein of (improve) at least 6.5 times.With the combination of mycobacterium bovis BCG
According to term " with the combination of mycobacterium bovis BCG ", it is understood as that co-administered any one mycobacterium bovis BCG bacterial strain together with the nucleic acid fragment of one or more fused polypeptide defined above or one or more these fused polypeptide of encoding, described mycobacterium bovis BCG bacterial strain comprises Pasteur (Pasteur), Phipps, Frappier, Connaught, Tice, Denmark, Ge Lansu (Glaxo), Prague, Birkhaug, Sweden, Japan, Moreau and Russian strain, or simultaneously but in different positions or with different administrations, its quantity can cause the specific immune response that significantly improves in animal model or people or effectively protect.
The reinforcement of mycobacterium bovis BCG
According to term " reinforcement of mycobacterium bovis BCG ", it is understood to be in the arbitrary period after any one mycobacterium bovis BCG inoculation, give as defined above one or more fused polypeptide or their one or more nucleic acid fragment of encoding, its quantity can cause the specific immune response that significantly improves in animal model or people or effectively protect, wherein mycobacterium bovis BCG bacterial strain comprises Pasteur, Phipps, Frappier, Connaught, Tice, Denmark, Glaxo, Prague, Birkhaug, Sweden, Japan, Moreau and Russian strain.
Polypeptide
Be used as the polypeptide of the unit of fused polypeptide of the present invention preferably from the immunogenic polypeptide of mycobacterium tuberculosis.This peptide species can such as based on the polypeptide deriving from Mycobacterium cell and/or mycobacterium tuberculosis culturing filtrate.Polypeptide that is that polypeptide is normally recombinated or synthesis, can be made up of immunogenic polypeptide, its immunogenic portion or can comprise extra sequence.Extra sequence can derive from natural antigen of mycobacterium tuberculosis or allos, and this sequence can but and nonessential there is immunogenicity.
According to term " fused polypeptide ", it is understood as that the immunogenic polypeptide from mycobacterium tuberculosis or its analog of two or more random alignment, wherein merges or do not merge the amino acid spacers (spacer) of one or more random lengths and sequence.
Word " polypeptide " has its common implication in the present invention, i.e. the amino acid chain of arbitrary length, comprises full-length proteins, oligopeptide, small peptide and fragment thereof and fused polypeptide, and wherein amino acid residue connects via the peptide bond of covalency.
Polypeptide can through chemical modification, comprise glycosylation, esterified (such as with the palmityl oxygen base butanimide described in Mowat etc. 1991 or by the lauroyl chloride compound described in Lustig etc. 1976 through chemistry esterified (lipidation)), comprise prothetic group or comprise extra aminoacid as histidine-tagged or signal peptide.
Each immunogenic polypeptide is by concrete aminoacid sign and by concrete nucleic acid sequence encoding.Also be within the scope of the present invention by restructuring or this sequence prepared of synthetic method and analog and mutant, this peptide sequence wherein in recombinant polypeptide has been substituted, has inserted, has added or has lacked one or more amino acid residue, but still has immunogenicity in described any biological detection here.
Replace preferably " conservative ", these conservatives replace as shown in the table.Aminoacid in identical group of the second hurdle, the aminoacid preferably in third column is gone together mutually, can replace each other.Aminoacid in third column is referred to by using single letter code.
Each polypeptide is by concrete nucleic acid sequence encoding.Analog and this nucleotide sequence being substituted, inserting, adding or lacking the modification of one or more nucleic acid are within the scope of the present invention.In codon uses, replace preferably reticent replacement, thus any one of amino acid sequence can not be caused to change, but can protein expression be improved by introducing.
Nucleic acid fragment
According to term " nucleic acid fragment " and " nucleotide sequence ", they are understood as that any one nucleic acid molecules, comprise DNA, RNA, LNA(and lock nucleic acid, locked nucleic acids), pentose nucleic acid (PNA), RNA, dsRNA and RNA-DNA hybridize chain.In addition the nucleic acid molecules of the nucleoside existed containing non-natural is comprised.Term comprises the nucleic acid molecules of any length depending on purposes, such as, from 10 to 10000 nucleotide.When nucleic acid molecules is used as a kind of pharmaceutical products, such as in DNA treatment, or for the preparation of according in the method for polypeptide of the present invention time, preferably use the molecule of coding at least one epi-position, its length from about 18 to about 1000 nucleotide, described molecule is optionally in insertion vector.When described nucleic acid molecules is used as probe, primer or antisense therapy, preferably use the molecule that length is 10-100 nucleotide.According to the present invention, other molecular length also can be used, such as there is the molecule of at least 12,15,21,24,27,30,33,36,39,42,50,60,70,80,90,100,200,300,400,500 or 1000 nucleotide (or nucleotide derivative), or there is the molecule of 10000,5000,4000,3000,2000,1000,700,500,400,300,200,100,50,40,30 or 20 nucleotide (or nucleotide derivative) at the most.
When using when being connected with hybridization conditions, term " strict " as defined in the literature, namely hybridization be temperature lower than the melting temperature Tm value described in the 11.45-11.49 pages in 1989 such as Sambrook at the most 15-20 DEG C time carry out.Preferably, condition is " high strict ", namely lower than melting temperature Tm value 5-10 DEG C.
Sequence iden
Term " sequence iden " refers to a kind of quantitative module of same degree between two substantially isometric aminoacid sequences or two substantially isometric nucleotide sequences.Two sequences relatively must be adjusted to has best potential identical probability with the truncate of the breach inserted or protein sequence end.Sequence iden can pass through formula (N
ref-N
dif) 100/N
refcalculate, wherein N
difthe sum of aniso-residue in latter two sequence of adjustment, wherein N
refthe quantity of the residue of one of two sequences.Therefore, DNA sequence AGTCAGTC and sequence A ATCAATC has the sequence iden (N of 75%
dif=2 and N
ref=8).It is the inconsistent of concrete (multiple) residue that breach be can be regarded as, and namely DNA sequence AGTGTC and DNA sequence AGTCAGTC will have the sequence iden (N of 75%
dif=2 and N
ref=8).Sequence iden can be calculated by blast program in addition, such as BLASTP program (Pearson W.R and D.J.Lipman(1988)) (www.ncbi.nlm.nih.gov/cgi-bin/BLAST).In an embodiment of the invention, calibration is with sequence comparison methods ClustalW, adopts the default parameter described for 1994 as Thompson J. etc. to carry out, and these can be obtained by network address http://www2.ebi.ac.uk/clustalw/.
The minimum percent of preferred sequence homogeneity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% and at least 99.5%.Preferably, relative to based on the immunogenic polypeptide unit of polypeptide deriving from mycobacterium tuberculosis, the number replacing, insert, add or lack one or more amino acid residue in fused polypeptide of the present invention is limited, namely there is 1,2,3,4,5,6,7,8,9,10 replacement at the most, 1,2,3,4,5,6,7,8,9,10 insertion at the most, 1,2,3,4,5,6,7,8,9,10 interpolation at the most, and 1,2,3,4,5,6,7,8,9,10 disappearance at the most.
Immunogenic portion
Polypeptide of the present invention comprises immunogenic portion as B cell or t cell epitope.
The immunogenic portion of immunogenic polypeptide is a part for polypeptide, and it causes the immunne response of the biological sample in animal or people and/or by arbitrary biotic experiment mensuration described herein.The immunogenic portion of polypeptide may be t cell epitope or B cell epi-position.Immunogenic portion can relate to the part of or some relatively little polypeptide, and they can be dispersed throughout on peptide sequence dispersedly or be arranged in the specific part of polypeptide.For some polypeptide epitopes, be even proved the full sequence (Ravn etc., 1999) of dispersion through polypeptide.
In order to the related T-cell epi-position be identified during identifying immunne response, likely use one " powerful (bruteforce) " method: because t cell epitope is linear, if systematically build the deletion mutant of polypeptide, so which region of display polypeptide is crucial for Immune discrimination by these mutants, such as, using object that these deletion mutants detect as IFN as described herein.Another kind method utilizes overlapping oligo-peptides for detecting MHC II class epi-position, and preferably synthesis has the epi-position that length such as 20 comes from the amino acid residue of polypeptide.These peptides can be determined in biological analysis (such as IFN described herein detects), and some in them will produce positive reaction (being therefore immunogenic), and this reaction is the evidence that there is t cell epitope in peptide.In order to find MHC I class epi-position, predict those will in conjunction with peptide (Stryhn etc., 1996), synthesis is afterwards prepared these peptides and is tested in relevant biological analysis IFN as described herein detects.Peptide preferably has the length that such as 8 to 11 derive from the amino acid residue of polypeptide.What within 1998, can be described by such as Harboe etc. determines B cell epi-position by analyzing the identification of B cell to the overlapping peptide covering polypeptide of interest.
The immunogenic portion of polypeptide can be identified by the major part of heritability heterogen crowd (altofrequency) or fraction (low frequency).But in addition, some immunogenic portion induce high immunne response (dominant), other then induce lower still effectively react (accounting for second advantage).That altofrequency >< low frequency can combine to the MHC molecule (HLA type) extensively distributed and even combined by multiple MHC molecule immunogenic portion relevant (Kilgus etc., 1991; Sinigaglia etc., 1998).
Analog
The common trait of fused polypeptide of the present invention is as illustrational in embodiment, and they can induce immune response.Certainly, by replacement, insert, add or lack the analog of the fused polypeptide of the present invention prepared also within the scope of the invention, described analog has immunogenicity equally through any detection assay described herein.
Substantially purification
In current context, term " substantially the polypeptide of purification " refers to polypeptide product, its comprise 5% weight at the most with described polypeptide natively or recombinate or synthesize produce in other peptide material of being associated (other peptide material of lower percent is preferred, such as at the most 4%, at the most 3%, at the most 2%, at the most 1% and at the most 0.5%).Preferably the polypeptide of purification is at least 96% purification substantially, and namely polypeptide accounts for 96% of the total peptide material weight be present in goods, and preferably higher percent, such as at least 97%, at least 98%, at least 99%, at least 99.25%, at least 99.5% and at least 99.75%.Particularly preferably polypeptide is " substantially pure form ", and namely polypeptide is substantially free of other antigen any of natural affiliation with it, does not namely contain other antigen any from the antibacterial belonging to tuberculosis compound group or virulent mycobacteria.This can realize by preparing polypeptide through recombination method in non-branch bacillus host cell, the same as will be described in detail, or by known solid phase or liquid phase peptide symthesis method improvement on synthesis, the method such as described by Merrifield or its derivative method, and by utilizing suitable purification step well known to those skilled in the art to realize.
Virulent mycobacteria, the individuality of current infection and the individuality of immunity
According to term " virulent mycobacteria ", it is understood as that the antibacterial that can cause tuberculosis disease in animal or people.The example of virulent mycobacteria is as mycobacterium tuberculosis, mycobacterium africanum, bacillus tuberculosis bovis, Mycobacterium leprae or mycobacterium buruli.The example of relevant animal is as cattle, didelphid, badger, Babalus bubalis L., lion, Dara (kurus) and kangaroo.
For " the current animal or human infecting virulent mycobacteria ", its be understood as that by cultivate or microscope confirm infected the individuality of virulent mycobacteria, and/or clinically diagnosis have TB and the individuality that anti-TB chemotherapy is responded.Cultivation, microscopy and clinical diagnosis TB are known by any person skilled in the art.
The individuality of immunity is defined by removing or controlling the infection of virulent mycobacteria or received the human or animal of mycobacterium bovis BCG inoculation.
Immunogenicity
Immunogenic polypeptide is defined by the polypeptide of induce immune response.Immunne response can one of by the following method monitoring:
External cell effect by the relevant cell factor from lymphocyte release such as IFN-(determine, described cell separation from current or infectd animal or the people of virulent mycobacteria in the past, or is determined by the propagation detecting these T cell.Induction is by adding polypeptide or immunogenic portion to containing 1x10
5individual cell is to 3x10
5carry out in the suspension of individual cell per well.Cell separation autoblood, spleen, liver or lung, immunogenic portion to the concentration of adding polypeptide or polypeptide is no more than 20, and (g/ml suspension stimulates two to five days.In order to monitor cell proliferation, the radiolabeled thymidine pulse of cell, after incubation 16-22 hour, detects propagation by liquid scintillation counting.Positive reaction is greater than the reaction that background value adds two standard deviations.IFN-(can be determined by the ELISA method known by person skilled in the art by release.Positive reaction is greater than the reaction that background value adds two standard deviations.When monitoring immunne response to polypeptide, except IFN-(, other cytokine is also suitable for, as IL-12, TNF-(, IL-4, IL-5, IL-10, IL-6, TGF-(.For measure cytokine (exist as IFN-() another and sensitiveer method is ELISPOT method, be wherein separated autoblood, spleen, liver or lung cell be diluted to preferably 1 × 10
6cell/ml is to 4 × 10
6the concentration of cell/ml, exist concentration be no more than 20 (under the polypeptide of g/ml or the immunogenic portion of polypeptide, incubation 18-22 hour.1 × 10 is diluted to after cell suspension
6/ ml to 2x10
6/ ml transferring to be coated with anti-IFN-(on Maxisorp plate, incubation preferably 4 to 16 hours.(speckle that antibody and related substrates produce determines that (produce cell, this speckle can use anatomic microscope to count IFN-to have the second anti-IFN-by usage flag.Also the mRNA of the coding relevant cell factor of existence can be measured by round pcr.Usually one or more cytokines will by such as PCR, ELISPOT or ELISA measure.Person skilled in the art is appreciated that by the remarkable increase of any described cytokine total amount of specific polypeptid induction or reduces the immunocompetence that can be used to evaluate polypeptide.
Can also determine external cell effect by utilizing the T cell system deriving from immune body or m tuberculosis infection person, wherein said T cell system starts 10 to 20 days by the additional IL-2 of mycobacteria extracted from the work of bacterial cell or culturing filtrate.(polypeptide of g/ml suspension is to containing 1x10 to be no more than 20 by adding
5individual cell is to 3x10
5the T cell system of individual cell per well realizes induction, and incubation carries out two to six days.(ELISA that is released through of induction or other relevant cell factor detects IFN-.Can also monitor by using radiolabeled thymidine as above to detect cell proliferation the stimulation of T cell.For detecting for two kinds, positive reaction is greater than the reaction that background value adds two standard deviations.
To clinically or subclinical on infected virulent mycobacteria individuality carry out intradermal injection or topical application paster (patch) at the most 100 (after g polypeptide or immunogenic portion, if create the positive reaction of at least 5mm diameter after 72-96 hour in injection or application, then the cell effect in body can be confirmed as positive DTH and react.
By the specific antibody reaction in immune or infected individuality, the reaction of extracorporeal body fluid can be determined.The antibody existed can be measured by elisa technique or Western blotting, and wherein polypeptide or immunogenic portion are absorbed into the surface of nitrocellulose membrane or polystyrene.Serum is preferably with PBS dilution, and dilution ratio from 1:10 to 1:100, and is added on absorbed polypeptide, incubation 1 to 12 hours.By utilizing the second antibody of labelling, as by ELISA after measured this specific marker whether exist and just can determine whether there is specific antibodies, wherein positive reaction is greater than the reaction that background value adds two standard deviations, or alternatively by the existence of specific antibodies can be determined by visual response in Western blotting.
After inoculating with the polypeptide be present in adjuvant or DNA vaccination, another relevant parameter measures the protection of inducing in animal model.The animal model be applicable to comprises primates that the infection through virulent mycobacteria excites, Cavia porcellus or Mus.The reading of protection of induction can be in target organ relative to not inoculating bacterial load that animal reduces, relative to not inoculating time-to-live that animal extends and relative to not inoculating the loss in weight or pathology that animal reduces.
Preparation method
The DNA sequence of usual fused polypeptide of the present invention, this fused polypeptide of encoding can use any one in various method to prepare.
Fused polypeptide can use the DNA sequence of this polypeptide of coding to carry out restructuring preparation, and wherein DNA sequence to be inserted in expression vector and to express in applicable host.The example of host cell is escherichia coli.Have and be less than about 100 aminoacid and be usually less than 50 amino acid whose fused polypeptide and can also be prepared by technology synthesis well-known to those skilled in the art, the such as commercially available solid phase technique supplied, this technology is sequentially added into aminoacid on the amino acid chain of growth.
Fused polypeptide can also be prepared by adding fusion partner, can obtain the superior characteristic of polypeptide of the present invention by the method.Such as, those can promote when recombinating preparation polypeptide export (export), what can promote peptide purification is all interested with improving the immunogenic fusion partner of polypeptide of the present invention.The present invention is particularly including containing having merged two or more based on the fused polypeptide of immunogenic polypeptide deriving from mycobacterium tuberculosis polypeptides.
Other can improve the immunogenic fusion partner of product cytokine, such as IFN-γ, IL-2 and IL-12.In order to promote to express and/or purification, fusion partner can such as, be bacterial pilin (fimbrial protein), such as pili component pilin (pilin) and papA; Protein A; ZZ-peptide (ZZ-fusogenic peptide is sold by Pharmacia Corp of Sweden (Pharmacia)); Maltose-binding protein; Glutathion (gluthatione) S-transferring enzyme; (-tilactase or poly-histidine.Fusion rotein can by recombinant production in host cell is as escherichia coli, and between different fusion partners, form a join domain is also feasible.The join domain be between such as independent immunogenic polypeptide unit can comprise 1,2,3,4,5,6,7,8,9 or 10 aminoacid.
Interested fused polypeptide, by esterified polypeptide of the present invention, makes immunogenic polypeptide be present in a suitable manner in immune system like this.This effect knows from the vaccine based on Borrelia (Borrelia burgdorferi) OspA polypeptide such as described in WO96/40718A or based on the vaccine (Cote-Sierra J, 1998) of Pseudomonas aeruginosa (Pseudomonas aeruginosa) OprI lipoprotein.Another probability merges known signal sequence and N-terminal cysteine at immunogenic polypeptide N-terminal.When preparing in the production host be applicable to, this fusion causes esterified at N-terminal cysteine place of immunogenicity fused polypeptide.
Vaccine
An importance of the present invention is the vaccine combination about comprising fused polypeptide of the present invention.In order to ensure the optimum performance of this vaccine combination, preferably it comprises in immunology and acceptable carrier, inert matter or adjuvant on pharmacopedics.
For not vaccinated animal, can by the effective vaccine containing fused polypeptide of the present invention of animal identification, to the bacterial load reduced in the animal model that virulent mycobacteria excites in target organ be made, and extend the time-to-live and/or reduce the loss in weight or pathological conditions.
The carrier be applicable to is selected from the group that polymer forms, (multiple) polypeptide is attached on this polymer through hydrophobic noncovalent interaction, such as plastics, as polystyrene, or (multiple) polypeptid covalence is attached on this polymer, as polysaccharide or polypeptide shellfish (keyhole limpet) hemocyanin as blue or green in bovine serum albumin, ovalbumin or keyhole.The inert matter be applicable to is selected from the group that diluent and suspending agent form.Adjuvant is preferably selected from following formed group: dimethyldioctadecylammonium base ammonium bromide (dimethyloctadecylammonium bromide) (DDA), dimethyl two octadecylene base ammonium bromide (dimethyloctadecenylammonium bromide, DODAC), Quil A, poly-I:C, aluminium hydroxide, incomplete Freund's adjuvant, IFN-(, IL-2, IL-12, monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), trehalose two behenate (dibehenate) and muramyldipeptide (MDP) or mycobacterial lipids extract, especially the extract of non-polar lipid disclosed in PCT/DK2004/000488.
To comprise polypeptide be that this area is usual as the preparation of the vaccine of active component known, as US Patent No. 4, and 608,251,4,601,903,4,599,231 and 4,599, exemplify in 230, they are all introduced into herein as a reference.
Other method for obtaining vaccine adjuvant effect comprises use preparation, such as use aluminium hydroxide or phosphate (phosphate) (Alumen), the synthetic polymer (carbopol (carbopol Carbopol)) of sugar, protein coacervation in vaccine is made by heat treatment, by the cohesion to albuminous (Fab) antibody reactivation via pepsin, mix mutually with the lipopolysaccharide components of bacterial cell as Cryptosporidum parvum (C.parvum) or endotoxin or gram negative bacteria, in a physiologically acceptable oils inert matter as carry out in mannide (mannide) monoleate (AracelA) emulsifying or with the 20% perfluocarbon emulsifying soln (FDA (Fluosol-DA)) as closed substitute (block substitute).May comprising of other uses immune regulator such as cytokine or synthesis IFN-γ inducer such as poly-I:C to be combined with above-mentioned adjuvant.
Realize the interested technology (being introduced into here as a reference) that may be use Gosselin etc. and describe for 1992 of another kind of adjuvant effect.In brief, related antigen such as antigen of the present invention can with the antibody conjugate of the Fc-receptor on anti-monocyte/macrophage (or antigen binding antibody fragment).
In order to improve BCG vaccine, one or more relevant antigen fused polypeptide as of the present invention in one or more can mixs with BCG before administration, inject simultaneously a kind ofly produce the better cooperative effect protected to obtain with BCG.Being keep BCG and the present invention merge the independence of (multiple) polypeptide but use simultaneously for realizing another interested probability of cooperative effect, using them in different positions or by different approach.
In order to strengthen the BCG vaccine of current use, can before BCG typically starts to weaken and even weaken, such as when BCG is postvaccinal 2,5,10,15,20,25,30,35,40,50,55,60,65 or 70 years, the antigen of being correlated with is one or more fused polypeptide of the present invention such as.Thereafter can every the interval administration of rule, as 1,2,3,4,5 or 10 year, altogether to 5 time.
Vaccine is used in a kind of mode consistent with dosage formulation, and the amount used will be such as play prevention or treat effectively act on and have immunogenicity.The amount used depends on that the experimenter for the treatment of is as caused the ability of the individual immunity system of immunne response and required degree of protection.The dosage range be applicable to has the order of magnitude of each vaccination containing a few hectogamma fused polypeptide of the present invention, preferably from about 0.1 μ g to the scope of 1000 μ g, such as, in the scope of about 1 μ g to 300 μ g, particularly in the scope of about 10 μ g to 100 μ g.The drug regimen being suitable for first administration and booster injection also can change, but representational be then carry out inoculating after first administration or other administration.
The mode used can change widely.Any conventional method for vaccine administration is all applicable.These comprise per os, nose or mucosal administration, with the solid form (such as pill, suppository or capsule) containing active component, or to be present in the form in the upper acceptable dispersant of physiology, such as spraying, powder or liquid, or by the mode of parenteral injection, such as subcutaneous, Intradermal or intramuscular or applied dermally.The dosage of vaccine depends on route of administration, and according to wanting the stature size age of prophylactic immunization people and lesser extent being wanted prophylactic immunization people to change.At present, most of vaccine is injected through intramuscular administration by pin, and this probably continues as standard route of administration.But the bacterin preparation that induction comprises Mucosal immunity is developed, and is generally sent by mouth or nose.The delivery system for mucosa immunity-inducing the most extensively studied at present comprises cholera toxin (CT) or its B subunit.When this albumen is present in administration in bacterin preparation, it enhance mucosa immunne response and induction of the generation of IgA.The advantage of mouth, nose vaccine is sent conveniently.But the toxin coming from the modification of other microorganism fungus kind has the toxicity of reduction remains immunostimulating ability, and the toxin that the heat-labile toxin from gram negative bacteria such as modified or staphylococcus produce all can be used for producing similar effect.These molecules are particularly suitable for mucosa delivery.
Vaccine such as carries out parenteral through subcutaneous or intramuscular injection through injection usually.Other preparation being suitable for other administering mode comprises suppository and oral formulations in some cases.For suppository, traditional binding agent and carrier can comprise such as poly alkylene glycol or triglyceride; This suppository can form the mixture from the active component containing 0.5% to 10% scope, preferred 1-2% scope.Oral formulations comprises such as normally used excipient, the mannitol of such as pharmaceutical grade, lactose, starch, magnesium stearate, saccharin sodium (sodium saccharine), cellulose, magnesium carbonate etc.These compositionss take the form of solution, suspension, tablet, pill, capsule, extended release preparation or powder, the active component advantageously containing 10-95%, preferred 25-70%.
In many cases, repeatedly vaccine must be used.Especially, mycobacterial infections that these vaccines determine with the infection and/or treatment that prevent virulent mycobacteria can be used or strengthen the situation that previous vaccination crosses the people of BCG.When vaccine is prevention infection administration, it prophylactically gave before the clinical sign or symptom appearance of the infection of definition.
Due to hereditary variation, different individualities can produce the immunne response of varying strength with phase homopolypeptide.Therefore, vaccine of the present invention can comprise several different fused polypeptide and/or polypeptide to improve immunne response.Vaccine can comprise polypeptide or their immunogenic portion of two or more fused polypeptide or hungry induction, wherein the antigen of all hunger inductions or fused polypeptide as defined above, or some (but and not all) polypeptide can derive from virulent mycobacteria.In the embodiment of the latter, the polypeptide not necessarily reaching above-mentioned fused polypeptide standard may play a role due to they self immunity or only serve as adjuvant.
Vaccine can comprise 1-20, as 2-20 or and even the different polypeptide of 3-20 kind or fused polypeptide, the polypeptide that such as 3-10 kind is different or fused polypeptide.
The invention still further relates to immune animal and comprise people to resist the method for TB caused by virulent mycobacteria, described method comprises and gives animal fused polypeptide of the present invention, or vaccine combination of the present invention as above, or above-described live vaccine.In a preferred embodiment at present, animal or people are immune bodies as defined above.
The invention still further relates to the method for the preparation of immunogenic composition of the present invention, described method comprise preparation, synthesize or be separated fused polypeptide of the present invention and this fused polypeptide is dissolved or dispersed in for vaccine medium in, and optionally add other antigen of mycobacterium tuberculosis and/or carrier, inert matter and/or adjuvant substance.
Nucleic acid fragment of the present invention can be used for the expression in vivo of immunogenic polypeptide, and namely this nucleic acid fragment can be used in so-called DNA vaccination, as Ulmer etc. 1993 comment, it is introduced into as a reference.
Be defined in the plasmid DNA of the vaccinated encode fusion polypeptide of DNA at structure and preparation, host strain can be used as escherichia coli.Then incubated overnight carries the host strain of this plasmid interested, utilizes and obtains plasmid DNA as Qiagen Giga-Plasmid column test kit (Qiagen, Santa Clarita, CA, the U.S.) comprises endotoxin removal step purification.Endotoxin must not be contained as the vaccinated plasmid DNA of DNA.
Therefore, the invention still further relates to the vaccine comprising nucleic acid fragment of the present invention, this vaccine causes animal to comprise the expression of immunogenic polypeptide in human body, after application of vaccine, the resistance strengthened in fact is given animal and is comprised people by the quantity of the polypeptide of expressing effectively, enhances the resistance lungy that they cause virulent mycobacteria.
By gene and the DNA fragmentation with the polypeptide of immunity moderation responsibility of encoding of conbined usage coding expression product, the effect of this DNA vaccination can be strengthened.
A kind of probability realizing the immunne response being used for effective active cell can by expressing relevant immunogenic polypeptide in non-pathogenic microorganism or virus.The most known example of this microorganism is bacillus tuberculosis bovis BCG, Salmonella and pseudomonas, and the example of virus is vaccinia virus and adenovirus.Therefore, another important aspect of the present invention strengthens the BCG vaccine of work available at present, wherein the DNA sequence of one or more fused polypeptide as defined above of the coding of one or more copy introduced in the genome of microorganism by ad hoc fashion, described ad hoc fashion makes microorganism be able to fused polypeptide described in expression and secretion.The introducing of the nucleotide sequence of the present invention of expecting more than a copy enhances immunne response.
Another probability is that coding is integrated into attenuated virus as (Rolph etc., 1997) in vaccinia virus or adenovirus according to the DNA of fused polypeptide of the present invention.In the Cytoplasm that recombined vaccinia virus can enter infected host cell or nucleus, expection thus interested fused polypeptide can induce the protective immune response of anti-TB.
The invention still further relates to the purposes that fused polypeptide of the present invention or nucleic acid are used as treatment vaccine, as D.Lowry citing (Lowry etc., 1999) that describe in the literature.There is the antigen of therapeutic properties when being used as vaccine, can by reducing the order of severity of m tuberculosis infection in laboratory animal based on them or preventing the ability of the reactivation of previous infection to identify.The described compositions being used as treating vaccine can by method described above for the preparation of vaccine.
Accompanying drawing explanation
Fig. 1: come from ugandan HIV-feminine gender (TB+/HIV-) and the HIV-positive (TB+/HIV+) TB patient and from the normal healthy controls (matched group) of Denmark to the antibody response of Rv2660c.Line of cut (cut-off) is based on the ROC tracing analysis of 97% specificity levels.The sensitivity observed is presented at above the diagram of data;
The immunogenicity of Fig. 2: Rv2659c
With containing the DDA/MPL of Rv2659c with the interval of two weeks to organizing Fl(Balb/cxC57BL/6 more) mice carries out subcutaneous vaccination three times.Finally postvaccinal one week, passed through elisa assay by the INF-γ of 5 μ gg/ml Rv2659c post-stimulatory PBMCs secretion;
The protection that Fig. 3: Rv2659c induction Killing Mycobacterium Tuberculosis infects
With Rv2659c to how group Balb/c-C57BL/6 mice carries out subcutaneous vaccination totally three times with the interval of two weeks, latter 12 weeks of inoculation, evaluate Vaccine effectiveness by contrasting the non-immune minimizing counted with CFU in the mouse lung of BCG immunity.Result is represented as the meansigma methods of log10 colony-forming units (CFU) and each experimental group 6 mices in lung;
The immunogenicity of Fig. 4: Rv2660c
With the DDA/MPL containing restructuring Rv2660c albumen to Fl(Balb/cxC57BL/6) mice carries out subcutaneous vaccination three times, and inoculation is spaced apart two weeks.(A) in the end postvaccinal one week, the IFN-γ being carried out post-stimulatory PBMCs by the Rv2660c of ELISA to 0.2,1 or 5 μ gg/ml was analyzed.Finally postvaccinal three weeks, by ELISA, the INF-γ of the post-stimulatory splenocyte of restructuring Rv2660c (B) with 0.2,1 or 5 μ gg/ml is analyzed, be used to breeder reaction research through the post-stimulatory peripheral blood lymphocytes of restructuring Rv2660c (PBMCs) (C) of 0.2,1 or 5 μ gg/ml;
Fig. 5: the protection that the Killing Mycobacterium Tuberculosis of being induced by Rv2660c infects
With the interval of two weeks, subcutaneous vaccination is carried out three times to many group Balb/c-C57BL/6 mices with Rv2660c, after aerosol infects 6 weeks, by the CFU counting in lung and to compare with the Mus of BCG immunity with non-immune thus evaluate Vaccine effectiveness.Result is represented as log in lung
10the meansigma methods of colony-forming units (CFU) and each experimental group 6 Mus.Simultaneously by the bacillus calmette-guerin vaccine Danish1331(5x10 of single dose
4bacillus/mice) inject root of the tail (the base of the tail) as first subunit vaccine subcutaneous (s.c.) and, as positive control, do not give booster injection;
Fig. 6: Hybrid56, the immunogenicity of HyVac21 and HyVac28
With containing 5 microgram Ag85b-ESAT6-Rv2660c(H56), Ag85a-TB10.4-Rv2660c(H21) or DDA/TDB(LipoVac Ag85b-TB10.4-Rv2660c(H28)) every two weeks to organizing Fl(Balb/cxC57BL/6 more) Mus carries out subcutaneous vaccination totally three times.Latter one week of finally inoculation, after stimulating with fusion rotein Ag85b, TB10.4 or Rv2660c for immunity of 1 μ g/ml, is discharged (Fig. 6 A-C) by the IFN-γ of elisa assay PBMCs.
With Ag85b-ESAT6-Rv2660c finally postvaccinal three weeks, secreted at the recombinant Ag 85 B with 0.2,1 or 5 μ gg/ml, the post-stimulatory INF-γ of ESAT6 or Rv2660c by elisa assay splenocyte (D), PBMCs(E) be used to analyze the breeder reaction resisting 1 μ g/ml same antigen;
Fig. 7: the strong protection infected by Killing Mycobacterium Tuberculosis after Hybrid56 immunity
(A) 56(Hybrid56 is hybridized with Ag85B-ESAT6-Rv2660c()) with the interval of two weeks, subcutaneous vaccination is carried out totally three times to many group Balb/c-C57BL/6 Mus; at aerosol challenge after 2,6,12 and 24 weeks, evaluate Vaccine effectiveness by more non-immune with the CFU counting in the lung of the Mus of BCG immunity.(B) with Ag85b-ESAT6(Hybridl) or Ag85b-ESAT6-Rv2031c(Hybrid32) with the interval of two weeks, subcutaneous vaccination is carried out totally three times to many group B6 mices; after aerosol challenge 7,13,24,35 and 44 weeks, evaluate Vaccine effectiveness by CFU counting in the Mus lung of more non-immune and BCG immunity.Result is represented as log in lung
10the meansigma methods of colony-forming units (CFU) and each experimental group 6 Mus.Simultaneously by the bacillus calmette-guerin vaccine Danish1331(5x10 of single dose
4bacillus/Mus) inject root of the tail as positive control as the first subunit vaccine subcutaneous (s.c.), do not give booster injection;
Fig. 8: Kaplan-Meier survival curve (n=7), after the mycobacterium tuberculosis aerosol of low dosage excites, extends its time-to-live with Ag85b-ESAT6-Rv2660c fusion rotein to the immunity that Cavia porcellus carries out, and makes it the level reaching BCG immune animal;
The immunity of inducing and protection Fig. 9: Hybrid56(Ag85b-ESAT6-Rv2660c)
With the DDA/MPL containing restructuring Ag85b-ESAT6-Rv2660c (hybrid56) to Fl(Balb/cxC57BL/6) mice carries out subcutaneous vaccination three times, and inoculation interval time is two weeks.Latter ten weeks of finally inoculation, the IFN-γ of the splenocyte stimulated with Ag85b, ESAT6 or Rv2660c of 0.2,1 or 5 μ g/ml by elisa assay secretes (as illustrated in figure 9 a).Latter ten weeks of inoculation, by the minimizing evaluation Vaccine effectiveness of CFU counting in the lung of the immune mouse relative to vehicle control.Result is represented as log in each experimental group 12 Mus lungs
10colony-forming units (CFU) (Fig. 9 B).
Embodiment
Materials and methods
Animal
Female the C57BL/6xBalb/C F1 or the C57BL/6 mice that do not have special pathogen, in 8 to 16 ages in week, available from the Bomholtegaard of Denmark, be used to the analysis of immunne response and the research of protection, protected by CFU analyzing evaluation.Infection research carries out in the BSL3 equipment of national serum institute (Statens Serum Institute).Animal is raised in the cage of isolation, for feedwater and arbitrary sterile food.All animal rest periods of 1 week (rest period) are given before starting experiment.The Ag85B-ESAT6(Hybrid1 of the antigen preparation restructuring of restructuring) prepare according to previously described method (Olsen, van Pinxteren etc., 2001).Briefly, His label protein is expressed in escherichia coli XL-1Blue, and carries out protein anions displacement chromatography with HiTrap Q post (Pharmacia (Pharmacia), Uppsala (Uppsala), Sweden) after Talon column purification.Before dilution and storing, with 25mMHEPES buffer (pH8.0)-0.15M NaCl-10% glycerol-0.01% polysorbas20 dialysis sample.
By previously described identical method (Skjot, Oettinger etc., 2000) the preparation restructuring Rv2660c for other little Mycobacterium proteins.Briefly, the Rv2660c gene of total length obtains from M. tuberculosis genes group DNA through pcr amplification, and the Rv2660c gene sub-clone of total length is entered expression plasmid pDestl7.Described recombiant protein is produced in escherichia coli Bl21blue, substantially according to previously described (Theisen, Vuust etc., 1995) but use and comprise the phosphate buffer of 8M carbamide, Ni+ post carries out purification by the method for Metal ion affinity chromatography, after purification, removes recombiant protein.
According to description, by the restructuring of some specificity, by Hybrid56(Ag85B-ESAT6-Rv2660c), Hybrid32(Ag85b-ESAT6-Rv2031c), HyVac21(Ag85a-TB10.4-Rv2660c) and HyVac28(Ag85b-TB10.4-Rv2660c) fusion rotein clone into expression vector pDestl7(Invitrogen) in.
After with IPTG induction, fusion rotein is expressed in coli strain BL21.After (B-PER, Sigma (the Sigma)) cracking of gentle detergent and supersound process, collect the inclusion body of all four kinds of fusion rotein.The inclusion body of washing to be dissolved in the 20mM NaOAc+8M carbamide of pH4.9 and by Q agarose gel (sepharose) post to catch endotoxin.The flow liquid collected dilutes in Bis-tris buffer+8M carbamide pH6.5, regulates pH to 6.5.Described albumen then by CM sepharose to catch impurity, on Q sepharose post, collect albumen afterwards.With bis-tris buffer (pH6.5)+3M urea washes pillar.With the albumen of NaCl elution of bound.Then cross polydextran gel (Sephadex) post, buffer uses tris-HCl and 10% glycerol of 25mM pH8 instead.
Mankind's identification-serology
Before for ELISA, by adding E. coli extract (S3761, Promega, the Madison (Madison) of 20 μ l, WI) to the blood serum sample of 200 μ l, also then at room temperature mix incubation 4 hours, whole serum is depleted by cross-reactive antibody.After centrifugal (10.000xg, 10 minutes), in supernatant, add 0.05% Hydrazoic acid,sodium salt.Described ELISA carries out according to following, 96 hole Maxisorp(Nunc, Roskilde, Denmark) the microtitration plate 1.0 every holes of μ g/ml(100 μ l) containing antigen carbonate-bicarbonate buffer (pH9.6) 4 DEG C bag spent the night.Then with containing the PBS(PBS-T of 0.05%Tween20) wash plate 3 times.With 1:100 with containing 0.2% polysorbas20 and 1.0%(wt/vol) the PBS(dilution buffer of bovine serum albumin) dilute serum sample, the dilute serum of 0.1ml adds in hole pairs, in duplicate, at room temperature incubation one hour.After washing 3x with PBS-T, in plate, add peroxidase-conjugated rabbit anti-human Ig(P212, DAKO, Glostrup that 100 μ l are diluted by dilution buffer with 1:8000, Denmark), incubation 1 hour.Plate PBS-T washs 3 times, with N-tetramethyl biphenyl substrate (TMB plus, Kem-En-Tec, * * *, Denmark) incubation 30 minutes, by adding 1M H
2sO
4cessation reaction.Then optical density (the OD at 405nm place is measured
405).Vaccine preparation and immune step
With 5 microgram recombiant vaccine (Rv2659c, Rv2660c, Hybrid56, HyVac21, HyVac28 or Hybrid32) immune mouses; recombiant vaccine is with 25 μ g monophosphoryl lipid A (MPL; Corixa; WA; the U.S.) carry out sending; the latter is at two dioctadecylammonium bromide (dioctadecylammonium bromide) (DDA; 250 μ g/ dosage; Eastman Kodak, Inc., Rochester; N.Y.) emulsified to cumulative volume 200 μ l in; as described recently (Olsen, van Pinxteren etc., 2001) are such.Described vaccine (0.2ml/ Mus) is injected three times at dorsal sc (s.c.) every two weeks.Simultaneously by bacillus calmette-guerin vaccine Danish1331(5 × 10 of single dose
4bacillus/mice) inject root of the tail as the first subunit vaccine subcutaneous (s.c.), do not give booster injection.The immunity excited in advance is evaluated by the blood lymphocytes of 5 and 7 weeks and the splenocyte of postvaccinal 7 weeks first after inoculation first.Count of bacteria in experimental infection and organ
In order to assess the level of protection, within 10 weeks after first immunisation, excite mice, or adopt the Glas-Col of calibration to suck contact system by aerosol mode, send the mycobacterium tuberculosis Erdman of about 100CFU to each lung.After 2,6,12 or 24 weeks, (Hybrid56) or 7,13,24,35 or 44 weeks rear (Hybrid32) puts to death mice, and lung and spleen are taken for count of bacteria.Described organ is evenly dispersed in sterile saline, and serial dilution thing is laid on the Middlebrook7H11 agar of the 2-thiophene-carboxylic acid hydrazides being supplemented with every milliliter of 2mg, optionally to suppress to test the growth of residual BCG in organ.After incubation 2 to 3 week, bacterium colony is counted at 37 DEG C.
Lymphocyte is cultivated
Organ enters complete RPMI(GIBCO, Grand Island, NY by segregation through pore stainless steel filtering net, containing 2mM glutamine, and 100U/ml penicillin 6-potassium and 100U/ml streptomycin sulfate, 10%FCS and 50mM2-ME) homogenized.Blood lymphocytes at the lympholyte(Cedarlane of density gradient, Hornby, Ontario (Ontario), Canada) upper purification.Merge cell of each group five mices and to cultivate in triplicate in round bottom microtiter well (96 holes, Nunc, Roskilde, Denmark), the RPMI1640 culture medium of volume 200 μ l is contained in each hole, containing 2x10 in this culture medium
5individual cell, is supplemented with 5x10
-5m2-mercaptoethanol, 1mM glutamine, Pen .-Strep 5%(volume/volume) hyclone.The working concentration scope of described antigen of mycobacterium is from 5 to 0.2mg/ml.Culture is at 37 DEG C, 10%CO
2middle incubation 3 days, (IFN-γ is by following MBP enzyme linked immuno-adsorbent assay (ELISA) for measuring IFN-γ for the supernatant then shifting out 100 μ l.
For the enzyme-linked immunosorbent assay (ELISA) of IFN-γ
According to the description (Mabtech of manufacturer, AB.Sweden), utilize the commercial reagents box being used for IFN-γ and measuring, the level that two sandwich (double sandwich) ELISA method is used to IFN-γ in the double volumetric solution to culture supernatant is carried out quantitatively.In sample, the concentration of IFN-γ uses the standard curve produced from restructuring IFN-γ (LifeTechnologies) to calculate, and result pg/ml represents.Difference between repeating hole is as one man less than 10% of described meansigma methods.
The effect evaluation of the experimental infection in guinea pig model and vaccine
Buy from the female Hartley Cavia porcellus of the outbreed of Charles River laboratory (North Wilmington, Mass.) or given 10 by Intradermal
3the BCG of CFU dosage once, or is given Ag85b-ESAT6 or Ag85b-ESAT6-Rv2660c tri-times containing the emulsifying in DDA/MPL of 20 μ g, and these three immunization interval are 3 weeks.Six weeks after third time immunity, use the device (Glas-Col, Terre Haute, Ind.) calibrated to send about 20 bacillus and enter in the lung of each Cavia porcellus and give aerosol MTB and excite.The time-to-live of infected Cavia porcellus is determined by observing the change of food consumption aspect on animal basis every day, dyspneic evidence and Behavioral change.In addition, based on weekly, animal is weighed, until the super continuous decrease that observed the body weight showing disease in a few days.
Embodiment 1
The mankind of hungry inducing antigen are identified
In one group that provides at WHO tuberculosis Sample Storehouse (Tuberculosis Specimen Bank) the lung TB patient from Uganda (Uganda), the mankind of evaluation Rv2660c identify situation.Comprising the patient (respectively, N=94 and N=73) with negative and positive HIV feature.Matched group is by 100 healthy forming at the donor of Denmark's inhabitation, and the BCG coverage of estimation is greater than 90%.
Microtitration plate is coated with 1.0 μ g/ml(every hole 100 μ l) Rv2660c albumen and with the blood serum sample incubation that 100x dilutes, use the anti-human Ig of peroxidase-conjugated rabbit and tetramethyl biphenyl as substrate colour developing (result is shown in Figure 1).
Conclusion
In this study, the identification of hungry induced protein is tested.Based on from matched group with 97% the line of cut (cutoff) determined of sensitivity, likely confirm 45% HIV-case and 61% HIV+ case in TB infect.Experiment clearly demonstrates between MTB infection period, and RV2660c albumen is expressed and by immune system recognition.
Embodiment 2
The immunogenicity of antigen (Rv2659c) and the prevention of reactivation of hungry induction is given by contacting rear (post-exposure)
Mice by m tuberculosis infection and by antibiotics process to reduce bacterial load (burden), and enters and has the latent infection stage of bacterial load close to detection level.The incubation period of infecting, every two weeks to the adjuvant (as DDA/MPL) totally three time of mouse inoculation containing Rv2659c.Finally postvaccinal one week, secrete (Fig. 2) by the post-stimulatory INF-γ of elisa assay hemocyte Rv2659c.The albumen Rv2659c of hungry induction induces the ability of the protection of Killing Mycobacterium Tuberculosis reactivation
Many groups have the mice of the mycobacterium tuberculosis of hiding to be carried out subcutaneous vaccination totally three times by every two weeks with the Rv2659c be formulated in adjuvant (as DDA/MPL), evaluate Vaccine effectiveness according to relative to the minimizing of colony-forming units (CFU) in nonvaccinated (latent infection) mice, lung and spleen.The protection of anti-reactivation is evaluated for three months after vaccination.Relative to the mice (Fig. 3) of the not immune latent infection of reactivation, Rv2659c induces Pulmonary bacterial level to reduce 3 to 90 times.In order to evaluate the impact of Rv2659c inoculation on the pathology that latent infection mice likely occurs, from the inoculated mice of latent infection, take out lung tissue for histopathology.Obvious caseous necrosis, fibrosis or mineralization do not detected at injury region, do not find the infiltration of the inflammatory cell increased yet.
Conclusion
In this study, the potentiality of albumen Rv2659c as treatment vaccine of hungry induction are tested.When the two dioctadecylammonium bromide-monophosphoryl lipid A of adjuvant combination dimethyl containing Rv2659c albumen gives mice, induce/strengthen strong immunne response.Immunization causes bacterial load in lung to reduce 0.5-1.0log.Thus, after our research indicates contact, vaccination reduces or is delayed the reactivation of mycobacterium tuberculosis and does not cause the immunopathology symptom of pulmonary.
Embodiment 3
The immunogenicity of the anti-aerosol m tuberculosis infection that the antigen Rv2660c induced by hunger induces and protectiveness
Every two weeks to the adjuvant (as DDA/MPL) totally three time of mouse inoculation containing Rv2660c.Finally postvaccinal one week, the INF-γ after hemocyte is stimulated to secrete (Fig. 4 A) by elisa assay Rv2660c.Finally postvaccinal three weeks, splenocyte is used to measure was secreted (Fig. 4 B) by the post-stimulatory IFN-γ of Rv2660c, and hemocyte is then for measuring the breeder reaction (Fig. 4 C) of antigenic specificity.
Many groups are carried out the mice of subcutaneous vaccination three times by every two weeks with the Rv2659c be formulated in adjuvant (as DDA/MPL); excited by with the aerosol challenge containing mycobacterium tuberculosis, its Vaccine effectiveness evaluates from the minimizing of the colony-forming units relative to non-Mice Inoculated (CFU) of lung by being separated.Evaluation protection in 12 weeks after vaccination.Relative to not immune infected mice, Rv2660c induces Pulmonary bacterial level to reduce about 0.5log (10) (Fig. 5).
Conclusion
In this study, the albumen Rv2660c of hungry induction is tested as the potentiality of vaccine antigen.When the two dioctadecylammonium bromide-monophosphoryl lipid A of adjuvant combination dimethyl containing Rv2660c albumen gives mice, it is induction of strong immunne response.Immunity causes bacterial load in lung to reduce about 0.5log (10).
Embodiment 4
The Antigen Fusion of hungry induction forms preventative vaccine (vaccine of many phases)
By the immunne response after three kinds of fusion protein immunizations
With the adjuvant (as DDA/MPL) containing fused polypeptide Hybrid56, HyVac21 or HyVac28, subcutaneous vaccination twice was carried out to many group mices every two weeks.Latter one week of finally inoculation, analyzes the post-stimulatory IFN-γ of the one-component of hemocyte in 1 μ g/ml fusion rotein or fusion rotein and secretes (Fig. 6 A-C).After finally inoculating with Hybrid56 three weeks, splenocyte, after the one-component of the described fusion rotein with 0.2,1 or 5 μ g/ml stimulates, measured IFN-γ secretion (as Fig. 6 D) by ELISA.The breeder reaction of antigenic specificity in hemocyte was measured (Fig. 6 E) in the end postvaccinal three weeks.
The ability that three kinds of fused polypeptide induce Killing Mycobacterium Tuberculosis to infect in mice
Many groups mice is carried out subcutaneous vaccination three times by every two weeks with Hybridl, Hybrid56 and Hybrid32 of being formulated in adjuvant (as DDA/MPL); after aerosol challenge, by evaluating Vaccine effectiveness relative to the minimizing of colony-forming units (CFU) in the lung of natural (nonvaccinated) mice and spleen.Simultaneously by the bacillus calmette-guerin vaccine Danish1331(5x10 of single dose
4bacillus/mice) injected root of the tail by as the first subunit vaccine subcutaneous (s.c.), as the positive control (Fig. 7 A and Fig. 7 B) for the protection of research.Polypeptide Hybrid56(Ag85b-ESAT6-Rv2660c in Cavia porcellus) protective capability of anti-aerosol mycobacterium tuberculosis
Every two weeks, subcutaneous vaccination is carried out three times to many group Cavia porcelluss with the adjuvant (as DDA/MPL) containing fused polypeptide, every animal is weighed Primary Evaluation Vaccine effectiveness based on weekly.Simultaneously by the bacillus calmette-guerin vaccine Danish1331(5x10 of single dose
4bacillus/mice) injected by as the first subunit vaccine Intradermal (i.d.), as the positive control for the protection of research.Result is the survival curve shown in Fig. 8.
Conclusion
In this study, the immune potentiality of three kinds of fusion rotein (Hybrid56, HyVac21 and HyVac28) are studied.When giving mice containing the adjuvant combination DDA/MPL of fusion rotein, three kinds of single protein components, all induction of strong dose dependent immunne response, show their potentiality as vaccine of many phases.The immunne response of the Hybrid56 induction selected as an example is along with high-caliber protective immunity; this protective immunity constantly strengthens in time, and the level reached is obviously on the level of protection that can reach with the MTB vaccination of this standard of bacillus tuberculosis bovis BCG.And, similar replacement Rv2660c containing the potential antigen Rv2031c(Ag85b-ESAT6-Rv2031c of standard MTB) Unit three (triple) fusion rotein, its not display protection improved in time.Finally, in the guinea pig model of more (succeptibel) of susceptible, reinforcement is obtained to the high-caliber protection of Hybrid56.
Embodiment 5
The activity of the hungry inducing antigen merged the and contact preventative vaccine (vaccine of many phases) that afterwards (therapeutic) use
Mice by m tuberculosis infection and by antibiotics process to reduce bacterial load, and enters the latent infection stage of low bacterial load.The latency stage infected, every two weeks to the adjuvant (as DDA/MPL) totally three time of mouse inoculation containing fused polypeptide.Latter 15 weeks of finally inoculation, measures hemocyte by ELISA and secretes (Fig. 9 A) through the post-stimulatory IFN-γ of the one-component of fusion rotein described in 0.2,1 or 5 μ g/ml.The protective capability of fused polypeptide induction Killing Mycobacterium Tuberculosis reactivation
Many groups have the mice of the mycobacterium tuberculosis of hiding to be carried out subcutaneous vaccination totally three times by every two weeks by the fused polypeptide be prepared in adjuvant (as DDA/MPL), evaluate Vaccine effectiveness according to the minimizing of colony-forming units (CFU) in the lung relative to nonvaccinated (latent infection) mice.Protection evaluation in three months after vaccination of anti-reactivation.Fused polypeptide, induction of the reactivation obviously reduced, makes Pulmonary bacterial level decrease (Fig. 9 B) relative to the non-immune latent infection mice of reactivation.
Conclusion
In this study, based on antigen Rv2660c, ESAT6(Rv3875) and antigen 85B(Rv1886c) fusion rotein tuberculosis subunit vaccine as treatment vaccine potentiality studied.When the two dioctadecylammonium bromide-monophosphoryl lipid A of adjuvant combination dimethyl containing fusion rotein gives mice, induce/strengthen strong immunne response.In the latent infection phase of reactivation, immunization result in the minimizing of bacterial load in lung.Therefore our research shows the antigen inoculation immunity of the hunger induction contacting rear fusion, and this preventative vaccine (vaccine of many phases) reduces or be delayed the reactivation of mycobacterium tuberculosis.
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Claims (9)
1. immunogenic composition, vaccine or a pharmaceutical composition, it comprises: the B-cell of polypeptide Rv1284 or polypeptide Rv1284 or T-cell epitope.
2. immunogenic composition according to claim 1, vaccine or pharmaceutical composition, for the purposes that prevents disease, therapeutic use, vaccine of many phases, or for strengthening the immunity in first BCG (Bacille Calmette-Guerin) vaccination.
3. the immunogenic composition according to any one of claim 1 ~ 2, vaccine or pharmaceutical composition, it is intradermal administration, percutaneous dosing, subcutaneous administration, intramuscular administration or mucosa delivery.
4. the immunogenic composition according to any one of claims 1 to 3, vaccine or pharmaceutical composition are for the preparation of the purposes that prevents disease, therapeutic use or aforementioned both, the medicine of vaccine of many phases or for strengthening the application in the medicine of the immunity of first BCG (Bacille Calmette-Guerin) vaccination.
5. the immunogenic composition according to any one of claims 1 to 3, vaccine or pharmaceutical composition for the preparation of the application for the treatment of in the medicine of the activity tuberculosis that caused by virulent mycobacteria or latent tuberculosis disease, or for the preparation of strengthening the application in the medicine of the immunity of first BCG (Bacille Calmette-Guerin) vaccination.
6. the application in the medicine of the infection for the preparation of prevention virulent mycobacteria of the immunogenic composition according to any one of claims 1 to 3, vaccine or pharmaceutical composition.
7. the application according to claim 5 or 6, wherein, described mycobacteria is selected from mycobacterium tuberculosis, Mycobacterium bovis, mycobacterium africanum, Mycobacterium leprae or mycobacterium buruli.
8. the application in the compositions for the preparation of the prophylactic immunization of anti-mycobacteria, booster shot, the inoculation of many phases or Therapeutic Vaccination of the immunogenic composition according to any one of claims 1 to 3, vaccine or pharmaceutical composition.
9. application according to claim 8, wherein, described mycobacteria is selected from mycobacterium tuberculosis, mycobacterium africanum, Mycobacterium bovis, Mycobacterium leprae or mycobacterium buruli.
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| AU2003242504A1 (en) * | 2002-07-13 | 2004-02-02 | Statens Serum Institut | Therapeutic tuberculosis vaccines |
| MX2009010800A (en) * | 2007-04-04 | 2010-01-29 | Infectious Disease Res Inst Id | Immunogenic compositions comprising mycobacterium tuberculosis polypeptides and fusions thereof. |
| JP5713672B2 (en) | 2007-04-12 | 2015-05-07 | ジェニファー ライター， | Tuberculosis vaccine and method of use thereof |
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| CN101969976A (en) | 2008-01-11 | 2011-02-09 | 美国政府健康与人类服务部秘书处 | Polypeptide vaccine and vaccination strategy against mycobacterium |
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| ES2602430T3 (en) * | 2008-07-25 | 2017-02-21 | Glaxosmithkline Biologicals S.A. | Polypeptides, polynucleotides and compositions for use in the treatment of latent tuberculosis |
| US9603919B2 (en) * | 2009-03-31 | 2017-03-28 | Japan As Represented By The Director-General Of National Institute Of Infectious Diseases | Method for prophylaxis of influenza using vaccine for intranasal administration |
| MX370744B (en) * | 2009-04-24 | 2019-12-20 | A tuberculosis tb vaccine to prevent reactivation. | |
| SG178144A1 (en) * | 2009-07-29 | 2012-03-29 | Bernd Helmut Adam Rehm | Polymer particles and uses thereof |
| MY173004A (en) | 2012-07-10 | 2019-12-18 | Transgene Sa | Mycobacterial antigen vaccine |
| RU2520737C1 (en) * | 2013-04-25 | 2014-06-27 | Федеральное государственное бюджетное учреждение "Научно-исследовательский институт эпидемиологии и микробиологии имени почётного академика Н.Ф. Гамалеи" Министерства здравоохранения Российской Федерации (ФГБУ "НИИЭМ им. Н.Ф. Гамалеи" Минздрава России) | METHOD OF PRODUCTION OF IMMUNOGENIC COMPOSITION BASED ON FUSION PROTEIN pESAT6-DBD AND DEXTRAN, RECOMBINANT PLASMID pESAT6-DBD, STRAIN OF Escherichia coli, CHIMERIC PROTEIN ESAT6-DBD AND THEIR APPLICATION |
| RU2520078C1 (en) * | 2013-04-25 | 2014-06-20 | Федеральное государственное бюджетное учреждение "Научно-исследовательский институт эпидемиологии и микробиологии имени почетного академика Н.Ф. Гамалеи" Министерства здравоохранения Российской Федерации (ФГБУ "НИИЭМ им. Н.Ф. Гамалеи" Минздрава России) | METHOD FOR OBTAINING IMMUNOGENIC COMPOSITION BASED ON Ag85A-DBD HYBRID PROTEIN AND DEXTRANE; pAg85A-DBD RECOMBINANT PLASMIDE; Escherichia coli [pREP4, pAg85A-DBD] STRAIN; Ag85A-DBD CHIMERIC PROTEIN |
| RU2539026C1 (en) * | 2013-06-11 | 2015-01-10 | Федеральное государственное бюджетное учреждение "Центральный научно-исследовательский институт туберкулеза" Российской академии медицинских наук | RECOMBINANT PLASMID pESAT6-CFP10-DBD, RECOMBINANT STRAIN Escherichia coli M15 [pREP4, pESAT6-CFP10-DBD], METHOD FOR PREPARING, IMMOBILISING, CONCENTRATING AND PURIFYING RECOMBINANT PROTEIN ESAT6-CFP10-DBD ON DEXTRAN, RECOMBINANT PROTEIN ESAT6-CFP10-DBD AND IMMUNOGENIC COMPOSITION CONTAINING PROTEIN ESAT6-CFP10-DBD |
| WO2014210018A1 (en) | 2013-06-25 | 2014-12-31 | Aeras Global Tb Vaccine Foundation | Tuberculosis compositions and methods of using the same |
| SG11201605595YA (en) * | 2014-01-09 | 2016-08-30 | Transgene Sa | Fusion of heterooligomeric mycobacterial antigens |
| EP3471763A4 (en) * | 2016-06-16 | 2020-04-29 | International AIDS Vaccine Initiative, Inc. | Tuberculosis compositions and methods of treating or preventing tuberculosis |
| CN107970444B (en) * | 2016-10-25 | 2022-03-01 | 中国人民解放军第三0九医院 | Composite adjuvant and vaccine containing same |
| EP3983011A1 (en) | 2019-06-14 | 2022-04-20 | Statens Serum Institut | Fusion proteins for tuberculosis vaccines |
| WO2022066926A1 (en) * | 2020-09-23 | 2022-03-31 | The General Hospital Corporation | Bcg vaccinations for prevention of covid-19 and other infectious diseases |
| KR20220079081A (en) * | 2020-12-04 | 2022-06-13 | 주식회사 제넥신 | Pharmaceutical composition for preventing or treating tuberculosis |
| WO2023156676A1 (en) | 2022-02-21 | 2023-08-24 | Statens Serum Institut | A novel cationic adjuvant composition |
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